Cytokines are secreted proteins that regulate cellular growth and differentiation. These factors are especially important in regulating immune and inflammatory responses, regulating lymphoid development and differentiation. Cytokines also have critical functions in regulating immune homeostasis, tolerance, and memory. Not surprisingly, cytokines are critical in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (IBD) and psoriasis. Understanding the molecular basis of cytokine action provides important insights into the pathogenesis of immune-mediated disease and offers new therapeutic targets. [unreadable] [unreadable] The first step in cytokine signaling is activation of receptor associated kinases, known as Janus kinases, or Jaks. Following the activation of Jaks by cytokines, the next step in signaling is the activation of a family of transcription factors called Stats (signal transducers and activators of transcription). We showed that mice completely lacking in Stat5A and Stat5B have major disruptions of T and B cell development. Stat5A/B is also critical for lymphoid stem cells and for CD8+ T cell survival. Thus, loss of Stat5A/B results in a SCID, similar to the primary immunodeficiency seen with Jak3 deficiency. [unreadable] [unreadable] T cells orchestrate immune responses through their production of cytokines, shaping the character of the response during host defense and in autoimmune settings. For more than a decade, two fates of CD4+ T cells were recognized: T helper 1 (Th1) and Th2 cells. In addition to these well-known subsets, additional fates are now recognized. Regulatory T cells (Treg cells) are an essential subset that maintains peripheral tolerance. Tregs are generated in the thymus from CD4+ T cells (natural Treg cells or nTregs) and can be induced in the periphery (iTreg cells). Both nTreg and iTreg cells express a transcription factor, forkhead box protein 3 (Foxp3). Deficiency of Foxp3 results in lethal autoimmunity in mice and humans; the disease in humans is termed IPEX. We found that deficiency of Stat5A/B in CD4+ T cells abrogated Treg development in vivo and blocked conversion of peripheral cells to become iTregs. To more carefully dissect the function of Stat5A/B in the immune system, we selectively deleted these related transcription factors CD4+ T cells to understand their role in T cell differentiation. We further found that Stat5A/B directly bind the Foxp3 gene, particularly in conserved sites in the first intron. CD4-cre, Stat5fl/fl mice also develop widespread autoimmune disease including IBD. In related studies, we found that Stat3 is a critical negative regulator of Foxp3 expression. [unreadable] [unreadable] An even more recently recognized subset of CD4+ T cells is cells that preferentially produce the cytokine IL-17 (Th17 cells). IL-17 is a major inflammatory cytokine, which appears to contribute to the pathogenesis of many autoimmune and autoinflammatory disorders including rheumatoid arthritis, spondyloarthropathies, multiple sclerosis and IBD. Of interest is that Th17 cells are thought to be developmentally related to regulatory T cells (Tregs) as both subsets can be induced from naive CD4+ T cells in the presence of transforming growth factor-beta (TGFb-1) in the context of different cytokines. Using CD4-Cre Stat3fl/fl mice, we showed that Stat3 is essential for Th17 differentiation. We further showed using chromatin immunoprecipitation that Stat3 binds to the Il17 gene. Conversely, we found that tissue-specific deletion of a negative regulator of Stat3 termed SOCS3 resulted in increased generation of Th17 cells and widespread autoimmune disease characterized by increased expression of IL-17 and IL-17-target genes. [unreadable] [unreadable] We extended these studies to examine the factors that govern Th17 in human CD4+ T cells. We provided evidence for a key role of IL-23 but also showed that human CD4+ T cells have much greater plasticity with respect to cytokine production. Additionally, through analysis of Jobs syndrome patients, who have dominant negative mutations of STAT3, we showed that Stat3 function is also essential for human Th17 differentiation. Although, T cells from Jobs syndrome patients were able to produce interferon gamma and other cytokines, they failed to produce IL-17. [unreadable] [unreadable] During the present year, we are extending our studies on the role of Stats in helper T cell differentiation by further investigating the role of Stat3 in IBD. In a T cell transfer model of colitis, we found that absence of Stat3 prevents the development of IBD. There appear to be several explanations underlying the reduction in autoimmune disease: absence of Stat3 is associated with fewer IL-17-producing cells, expansion and conversion of Tregs and reduced proliferation. We are currently investigating the Stat3 in these processes. Another cytokine purportedly produced by Th17 cells is IL-22. IL-22 is a complex cytokine with both pro- and anti-inflammatory effects. We are trying to define the relative importance of Stat3 with respect to IL-17 and IL-22. [unreadable] [unreadable] By performing microarrays, we found that Th17 cells express the cytokine IL-21. We found that IL-21 production was also dependent upon Stat3 and also found that IL-21 promotes IL-17 production; in this respect, the autocrine production of IL-17 by Th17 is analogous to other Th subset (e.g. Th2 cells and IL-4). [unreadable] [unreadable] Our microarray data also showed that Th17 cells express the orphan retinoid receptor Rorgt. In the absence of Stat3, the former transcription factor is poorly induced. We found that agonists of another class of retinoid receptor, retinoic acid, inhibited Th1, Th2 and Th17 differentiation. Retinoic acid downregulated expression of Rorgt but importantly, retinoic acid also upregulated Foxp3 and induced functional Tregs. Conversely, antagonist of retinoic acid receptor alpha (Rar alpha) blocked Foxp3 expression. Interestingly, immunosuppressive mucosal dendritic cells produce retinoic acid and this is now thought to be a mechanism by which they promote immune tolerance in the gut. [unreadable] [unreadable] We also studied the effect of another cytokine, IL-27. Lack of IL-27 is also associated with fatal, widespread autoimmune disease. One function of IL-27 is its role in potently inhibiting Th17 cells. This aspect of IL-27s effect is mediated by Stat1. In addition though, IL-27 has another important function namely to produce another anti-inflammatory cytokine, IL-10. We found that the ability of IL-27 to induce IL-10 is dependent upon Stat3. [unreadable] [unreadable] In related studies, we also found that IL-2, an important product of activated T cells, inhibited Th17 differentiation and enhanced Treg differentiation. We found that both of these effects were dependent upon Stat5. Conversely, blockade of IL-2 promoted Th17 differentiation and attenuated Treg differentiation. We found that in IL-2-deficient mice, which have systemic autoimmune disease, also have expansion of Th17 cells. IL-2 activates Stat5 and we found that Stat5-deficient T cells had increased tendency to become Th17 cells. In related studies, we found that IL-2 inhibited its own production in a Stat5-dependent manner. [unreadable] [unreadable] During this year, we are also studying the function of Stat5 in the initial phases of T cell receptor-mediated activation. In addition, we are assessing the function of Stat5 in Th2 differentiation and IL-4 production, and are further defining the inflammatory bowel disease evident in Stat5-deficient mice. This year, we have also initiated studies mapping genomewide chromatin modifications, examining the role of Stats in the process. Specifically, we analyzed histone modifications in Th1, Th2, Th17, and Treg cells by Chip-Seq technology. We have also assessed the importance of Stat4 in chromatin modifications in Th1 cells using Stat4-deficient mice and Chip-Seq technology.